Biology subjects

Shiwarski, D. J.

Publications and source records attributed to Shiwarski, D. J..

8 recordsLinked to original sources

bioRxiv2025

Development of an Open-source Low-cost Pressure Myography and Cardiac Flow Simulator, HemoLens, for Mechanical Characterization of Native and Engineered Blood Vessels

Pressure myography, the standard for assessing vascular mechanics and vasoreactivity, is costly ($40,000+), has low throughput, and is limited to static fluid flow. Here, we developed HemoLens, an open-source 3D-printed pressure myography system for [~]$700. HemoLens features compact micromanipulators, incremental in-line pressure control, physiological temperature regulation, and modular pulse pressure control between normotensive and hypertensive levels. HemoLens efficacy was demonstrated by delineation of physiological reactivity and pathological mechanical phenotypes using native mouse arteries and bioprinted acellular scaffolds. Wildtype vessels show greater distention (124.3 vs. 43.07 {micro}m) and increased dynamic compliance compared to diseased vessels. Small diameter (450 {micro}m) collagen-based artery-like scaffolds are FRESH bioprinted to mimic hypertensive vascular stiffening. Engineered hypertensive vessels demonstrate increased burst pressure (464 mmHg) and reduced dynamic compliance reminiscent of diseased arteries. Together, HemoLens lowers the barrier to entry in pressure myography research by serving as a comprehensive low-cost system for native and engineered vessel characterization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/651300v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@6b87d0org.highwire.dtl.DTLVardef@1a17b5eorg.highwire.dtl.DTLVardef@aa6056org.highwire.dtl.DTLVardef@19ac816_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

3D Bioprinting of Collagen-based Microfluidics for Engineering Fully-biologic Tissue Systems

Microfluidic and organ-on-a-chip devices have improved the physiologic and translational relevance of in vitro systems in applications ranging from disease modeling to drug discovery and pharmacology. However, current manufacturing approaches have limitations in terms of materials used, non-native mechanical properties, patterning of extracellular matrix (ECM) and cells in 3D, and remodeling by cells into more complex tissues. We present a method to 3D bioprint ECM and cells into microfluidic collagen-based high-resolution internally perfusable scaffolds (CHIPS) that address these limitations, expand design complexity, and simplify fabrication. Additionally, CHIPS enable size-dependent diffusion of molecules out of perfusable channels into the surrounding device to support cell migration and remodeling, formation of capillary-like networks, and integration of secretory cell types to form a glucose-responsive, insulin-secreting pancreatic-like microphysiological system. One-Sentence SummaryMulti-material FRESH 3D bioprinting of microfluidic CHIPS to generate fully biologic centimeter-scale and vascularized pancreatic-like tissue systems.

bioengineering↗

bioRxiv2022

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗

bioRxiv2021

In Situ Volumetric Imaging and Analysis of FRESH 3D Bioprinted Constructs Using Optical Coherence Tomography

As 3D bioprinting has grown as a fabrication technology, so too has the need for improved analytical methods to characterize these engineered constructs. This is especially challenging for soft tissues composed of hydrogels and cells as these materials readily deform, posing a barrier when trying to assess print fidelity and other properties non- destructively. Indeed, given the importance of structure-function relationships in most tissue types, establishing that the 3D architecture of the bioprinted construct matches its intended anatomic design is critical. Here we report development of a multimaterial bioprinting platform with integrated optical coherence tomography (OCT) for in situ volumetric imaging, error detection, and 3D reconstruction. While generally applicable to extrusion-based 3D bioprinting, we also report improvements to the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting process through new collagen bioink compositions, support bath optical clearing, and machine pathing. This enables high-fidelity 3D volumetric imaging with micron scale resolution over centimeter length scales, the ability to detect a range of print defect types within a 3D volume, and real-time imaging of the printing process at each print layer. These advances provide FRESH and other extrusion-based 3D bioprinting approaches with a comprehensive methodology for quality assessment that has been absent in the field to date, paving the way for translation of these engineered tissues to the clinic and ultimately to achieving regulatory approval. TeaserTransparent FRESH support bath enables in situ 3D volumetric imaging and validation of patient-derived tissue constructs.

bioengineering↗

bioRxiv2021

Kidney-Specific WNK1 Amplifies NCC Responsiveness to Potassium Imbalance

To maintain potassium homeostasis, the kidneys distal convoluted tubule (DCT) converts small changes in blood [K+] into robust effects on salt reabsorption. This process requires NaCl cotransporter (NCC) activation by WNK kinases. During hypokalemia, the Kidney-Specific WNK1 isoform (KS-WNK1) scaffolds the DCT-expressed WNK signaling pathway within biomolecular condensates of unknown function termed WNK bodies. Here, we show that KS-WNK1 amplifies the dynamic range of NCC activity in response to potassium imbalance, in part via WNK bodies. Targeted condensate disruption traps the WNK pathway, causing renal salt-wasting that is more pronounced in females. In humans, WNK bodies accumulate as plasma potassium falls below 4.0mmol/L, suggesting avid condensate-mediated salt reabsorption even when [K+] is low-normal. These data identify WNK bodies as signal amplifiers that mediate tubular potassium responsiveness, nephron sexual dimorphism, and blood pressure salt-sensitivity. Our results illustrate how condensate specialization can optimize a mammalian physiologic stress response that impacts human health.

physiology↗

bioRxiv2021

Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells

Cell injection is a common clinical approach for therapeutic delivery of cells into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site has generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix (ECM) that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These Monolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted Monolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in ECM dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness. One Sentence SummaryShrink-wrapped patches of endothelial cells can rapidly attach and integrate into an intact cornea endothelium when injected into the anterior chamber, increasing cell density.

bioengineering↗

bioRxiv2020

Dynamic Loading of Human Engineered Heart Tissue Enhances Contractile Function and Drives Desmosome-linked Disease Phenotype

The role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%. To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre- and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output. Single Sentence SummaryDevelopment of a dynamic mechanical loading platform to improve contractile function of engineered heart tissues and study cardiac disease progression.

bioengineering↗

bioRxiv2020

Fibronectin-Based Nanomechanical Biosensors to Map 3D Strains in Live Cells and Tissues

Mechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 {micro}m. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

bioengineering↗